Displacement Chromatography

12/30/2011

If you are purifying Monoclonal antibodies for therapeutic use, you cannot afford to ignore displacement chromatography. Below are three commonly asked questions about displacement.

1. Can displacement chromatography be used for the production scale purification of antibody (Is it an alternative to using Protein A)?

---Cation exchange displacement chromatography has been done on antibodies using our displacers. Displacement as alternative to Protein A work has been done by Lonza in Europe and a patent is filed.

2. What is the merit & cost cutting compared with the Protein A?

The price of Protein A is one expense that is less when using displacement but also the elimination of charge variants and the ability to load at higher levels translate into an overall cost savings. One of the key benefits of displacement chromatography is that displacement helps putting light into the science of mAb production. By detecting charge variants previously unknown in a substance, you can then determine whether these trace components found in a lab-scale process have the potential to impact the efficacy of your therapeutic when produced commercially. There is a huge risk elimination factor associated with this knowledge.

3. What data and real-world examples are available for scientists for further research on displacement?

Simply put, if you are purifying Monoclonal antibodies for therapeutic use, you cannot afford to ignore displacement chromatography using SACHEM EXPELL™-SP for protein purification or ISOLIS™-SP for trace component detection. It is the best approach.

08/08/2011

In their recently published paper Isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography, research scientists Taylor Zhang, Justin Bourret and Tony Cano state "The advent of quality by design (QbD) in the biotechnology industry has increased the need for the isolation and enrichment of specific variants for biotherapeutic products [1]." MAb variants may form during any part of the antibody manufacturing process and their identification and/or isolation is critical as any of them can potentially affect the efficacy and safety of the therapeutic, the authors point out and continue that although the isolation and analysis of specific variants is a common requirement and "essential for assessment of biophysical and biochemical properties such as in vitro (potency and binding) and in vivo (toxicity, pharmacokinetic, immunogenicity) activity,"[2] their characterization is challenging due to the fact that most charge variants exist only at trace levels. The scientists were able to more efficiently isolate mAb charge variants and increase thoughput 10x by supplementing their mAb characterization toolkit with displacement chromatography, "a powerful but unterutilized tool in the process development and characterization of complex biological therapeutics" [2].

06/13/2011

Charge-related heterogeneity is a common issue in nearly every monoclonal antibody process. It is clear that with the pharmaceutical industry shifting its focus to mAbs, there will be more and more types of heterogeneity uncovered.

A very nice review by Vlasak and Ionescu from Merck Laboratories was put together in 2008 and details quite a few of the currently well known charge variants and isolation by methods which exploit this charge variation (1). Briefly, while many of these variations resulted from modification at the COOH terminus, there were also conformational variants such as disulfide related variants, which, in a standard cation exchange chromatogram, would be very subtle and would run as a basic variant (2) on a cation exchange column. The acidic variants leading to conformational change may also show up due to loss of NH2 through N-terminal glutamate cyclization (3). While the main chromatographic approach to resolving or rather attempting to resolve these modifications is typically weak cation exchange chromatography, there are other lesser developed approaches such as hydrophobic interaction chromatography as well as reversed phase chromatography evaluating the antibody heavy and light chain fragments. (4).

An advantage of reversed phase approaches is the ability to couple the analysis directly to mass spectrometry. One common problem is that intact antibody heavy and light chains could possibly have these subtle variations masked in RP-MS analysis. Therefore, an additional step would be required to reduce the complexity such as enzymatic digestion prior to analysis or rather a proteomic approach. In the sum of things, these types of analysis, while very sophisticated, often lead to diminishing returns as there is relatively little material to analyze further once the modified protein has been isolated and in the event of digestion for mass spectrometry analysis, it simply would not be possible to analyze for binding properties or pharmacokinetic properties.

Genentech has published a very elegant study, which shows that it is possible to isolate these diverse variants in enough quantity and efficiency to enable pharmacokinetic analysis of intact antibody variants (5). Their approach was to use SACHEM’s powerful cation exchange displacement reagents applied to their own chromatography resins. Not only were they able to separate various charge variants but they were also able to isolate them in quantity and significantly higher efficiency than using standard elution phase approaches. Vlasak and Ionescu list many variants that can be resolved and studied further using the SACHEM method. (Dr. Pat McAtee)

03/31/2011

Heterogeneity in antibody production is a hot topic in the biological drug discovery business these days. It has long been known that production of antibodies in cell culture can lead to variation in antibody structure. However, it has yet to be shown that these variations truly have an effect in antibody structure and function in immunological assay or in circulating plasma. In a series of publications, Amgen scientists have conclusively demonstrated that circulating human IgG2 antibodies isolated from myeloma patients and normal serum appear to be a naturally occurring feature of antibodies belonging to the human igG2 subclass. (Wypych et. al 2008). In particular, they report the effects of disulfide bond heterogeneity, which can presumably affect antigen binding affinity and avidity and possibly affect the immune function at an organismal level. While binding activity assays of the human IgG2 monoclonal antibody did not appear to be effected, this was not the case in an accompanying study (Dillon et. al 2008). In this accompanying report, they noted that disulfide mediated heterogeneity was indeed able to affect structure and function, which was manifested in several cell-based assays.

While the group was able to partially purify the antibodies on cation exchange columns to pursue their functions, they noted that further studies were necessary to fully characterize these variations in antibody structure. However, purification of these isoforms can be somewhat daunting. This is where the SACHEM approach of purification of antibody variants using displacement chromatography to isolate and concentrate the charge variants can be very useful. In fact, this approach has been used to generate quantities of these variant antibodies for pharmacokinetic studies in animals (Khawli et. al. 2010). So for antibody groups who are looking for ways to conclusively nail antibody heterogeneity issues, displacement offers a unique and robust approach to solving these problems.

01/14/2011

Last week, we compared how substances emerge in the two chromatographic modes: displacement (as trapezoidal bands-the displacement train) and elution (as isolated, solvent-separated peaks). Today, let's have a look at the resolution of substances

In elution chromatography, for two substances to travel at different speeds and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. In order to achieve baseline separation of the peaks, operational simplicity and throughput are often sacrifices resulting in gradient elution and low column loadings.

Resolution of Substances: Displacement

The same interaction described for elution chromatography is also important to displacement. There is competition between the separating species for the sames site. The molecules that have the strongest attraction to the matrix bind and push the weaker species off to bind at a different site. This leads to sharpened adjaceing bands and better resolution int he displacement train.

The separation may be run at high loadings since there is no need to try to prevent component overlap.

01/06/2011

Displacement chromatography using ion-exchange matrices works great for mAb's, especially following an affinity step (protein A) or as the final polishing step. It is usually difficult to compare a finishing step (ion-exchange) with an affinity step (protein A) because they operate so differently. However, we know of cases where the advantages of displacement chromatography (IEC, HIC) together with a cleaner input stream render the earlier affinity step unnecessary.

In these cases, ion-exchange chromatography operated in displacement mode functions well as a capture step.

10/26/2010

As we travel along the path of innovation, a new product or technology (imagine the early beginnings of cell phones, the Internet) first is nearly impossible to do, then it is made possible. After being impractical to do/use, the technology is improved. As the cost of implementation decreases, the "innovative" technology becomes possible and accessible. Soon the availability and use of the "new" tools becomes expected and then required (i.e. incorporated in school curricula). Along this path of innovation, there is a sweet spot of entrance, of trying out new things for every organization or individual. Some of us are early adopters, others wait a little and then there are those, that may wait too long.

For each new technology, companies go through the process of determining their individual "sweet spot" of adapting to and traveling along the learning curve of incorporating a new technology into their processes. Where is your sweet spot of joining countless other R&D scientists at leading science companies who are exploring displacement chromatography?

What does this have to do with displacement chromatography? Read how Genentech utilized Displacement chromatography for mAb charge variant analysis to understand their processes and make them more effective.

09/09/2010

Identical twins have the same DNA, the exact same genes and the same DNA. Two people start out the same, but their appearance and their style can change the way they look. Our lifestyles and environment can change the way our genes are expressed, leading even identical twins to become distinct as they age.

Imagine this: one identical twin gets diagnosed with cancer, the other is perfectly healthy. Or here is another example, how can one twin develop a metabolic disorder whereas the other stays "in shape"? How can two genetically alike individuals be so similar and so different?

Some researchers have discovered a new bit of biology that can work with our genes or against it: Epigenetics. Epigenetics literally translates as “above the genome" [i.e. the epigenome is the control center or HQ of a cell - in very simple, technically not 100% correct terms]. Identical twins can be genetically identical, but may not share the same epigentical destiny, especially if their lifestyles differ. The epigenome acts similar to the software in a computer telling a cell how it should act; an epigone can turn on or off a certain genetic function. (If you want to learn more, watch this great introductory video by NOVA Science here.)

Epigenetics is a subtle modification that affects metabolism, malignancy, cognitive function, and even itchy, scratchy feet!

Once identified, compounds [and eventually drug therapies ] that target certain epigenetic functions can be developed, i.e. to control cancer or obesity cell function. This represents a significant step towards characterizing the epigenome with respect to the variation in the non-inherited phenotype.

08/30/2010

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants pose a challenge to demonstrating product comparability. Check out the report published in Landes Bioscience Journal "mAbs" (Vol 2 Issue 6) to read more on how Genetech gained further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity by utilizing cation exchange displacement chromatography to isolate the major charge forms of a recombinant humanized IgG1 and compare their in vitro properties and in vivo PK.

To access the full report (subscription based) and read how Genentech utilized displacement chromatography for mAb charge variant anlysis, please follow the link:

Need more reasons or supporting data to believe in displacement chromatography? Or do you want to join your fellow scientists and try out SACHEM's QuickStart™ Displacement Starter Kit™, an "all-inclusive" package that provides an introduction to displacement chromatography and opportunity to evaluate this tool for your processes?

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